Use of aminoadamantane compounds as immunoregulators

ABSTRACT

wherein R 1 , R 2 , R 3  and R 4  are selected independently from each other from —NR 5 R 6 , —NR 5 R 6 R 7   + , hydrogen, aryl or heteroaryl with up to 7 ring members, C 1 -C 20 -alkyl, C 1 -C 20 -alkenyl and C 1 -C 20 -alkinyl, wherein the alkyl, alkenyl and alkinyl residues can be branched, unbranched or cyclized and optionally substituted with halogen, aryl or heteroaryl with up to 7 ring members, with the proviso that at least one of the residues R 1 , R 2 , R 3  and R 4  are represented by —NR 5 R 6  or —NR 5 R 6 R 7 ; and 
     R 5 , R 6  and R 7  are selected independently from each other from hydrogen, aryl or heteroaryl with up to 7 ring members, C 1 -C 20 -alkyl, C 1 -C 20 -alkenyl, C 1 -C 20 -alkinyl, wherein the alkyl, alkenyl and alkinyl residues can be branched, unbranched or cyclized and optionally substituted with halogen, aryl or heteroaryl with up to 7 ring members, or R 5  and R 6 , together with the nitrogen atom, form a heterocyclic group with up to 7 ring members; 
     for the regulation of the activity of already activated neutrophils.

The invention relates to the use of aminoadamantane compounds asimmunoregulators, especially for the regulation and modulation ofalready activated neutrophils.

Neutrophils, a subclass of leukocytes, are involved in immune defenseand other reactions in the blood vessel system and bordering tissues(Roitt, I. M, Leitfaden der Immunologie, Steinkopf-Verlag Darmstadt, 2ndedition, 1984).

Neutrophils can be activated by exogenous substances, such as forexample cell wall components (a commercially obtainable product is“Zymosan”). In connection with this, reactive oxygen species are formedin the so-called respiratory burst.

FIG. 1 describes a simplified scheme of neutrophil stimulation: Afterbinding of a ligand (for example, Zymosan A) to a specific receptor onthe neutrophil membrane, guanosine triphosphate (GTP) is activated,which in turn activates the phosphatidyl-inositol-4,5-biphosphate(PIP₂)-specific phospholipase C (PLC). PLC catalyses the hydrolysis ofPIP₂ to inositol-1,4,5-triphosphate (IP₃) and diacylglycerol (DG). IP₃stimulates the release of Ca²+ ions from intracellular stores. Ca²⁺ions, together with DG, activate the enzyme protein kinase C (PC) whichphosphorylates numerous proteins and in this manner activates NADPHoxidase complex among others. NADPH oxidase catalyses in turn thereaction:

2O₂+NADPH→2O₂ ⁻+NADP⁺+H⁺

wherein the reactive oxygen species O₂ ⁻ results.

Although activated neutrophils are important for a functioning immunedefense, disease symptoms such as acute or chronic inflammations orother allergic reactions can result by overreactions caused especiallyby reactive oxygen species. On the other hand, neutrophil activity canbe too low or insufficient for a successful immune defense with ageneral immune deficiency such as for example with AIDS. Hence, a needexists, especially from the medical standpoint, to be able to influencethe regulative and/or modulating activity of neutrophils in vivo and invitro. In this connection, the neutrophil activity should be eitherincreased or decreased depending on the indication, i.e. should becontrollable at will.

Therefore, the object of the present invention is to make available anagent for the regulation and/or modulation of the activity ofneutrophils.

It has now been surprisingly found that certain aminoadamantanecompounds have a regulative and/or modulating effect on the activity ofneutrophils. The present invention relates to this finding. It isdecisive for this that the neutrophils have already been activated byother stimulants. The aminoadamantanes themselves do not demonstrate anyactivating effect on neutrophils.

The above object is accordingly solved by the use of aminoadamantanecompounds of formula (I)

wherein R₁, R₂, R₃ and R₄ are selected independently from each otherfrom —NR₅R₆, —NR₅R₆R₇ ⁺, hydrogen, aryl or heteroaryl with up to 7 ringmembers, C₁-C₂₀-alkyl, C₁-C₂₀-alkenyl and C₁-C₂₀-alkinyl, wherein thealkyl, alkenyl and alkinyl groups can be branched, unbranched orcyclized and optionally substituted with halogen, aryl or heteroarylwith up to 7 ring members, with the proviso that at least one of thegroups R₁, R₂, R₃ and R₄ are represented by —NR₅R₆ or —NR₅R₆R₇; and

R₅, R₆ and R₇ are selected independently from each other from hydrogen,aryl or heteroaryl with up to 7 ring members, C₁-C₂₀-alkyl,C₁-C₂₀-alkenyl, C₁-C₂₀-alkinyl, wherein the alkyl, alkenyl and alkinylgroups can be branched, unbranched or cyclized and optionallysubstituted with halogen, aryl or heteroaryl with up to 7 ring members,or R₅ and R₆, together with the nitrogen atom, form a heterocyclic groupwith up to 7 ring members; for the regulation of the activity of alreadyactivated neutrophils.

Aminoadamantane compounds of this type are known. Thus,1-aminoadamantanes are described in DE 22 19 256, DE 28 56 393, DE 22 32735, U.S. Pat. Nos. 3,450,761 or 4,122,193. The production of compoundsof formula (I) generally occurs by known methods, such as for examplethe alkylation of halogen adamantanes. Subsequent further halogenationand alkylation produce the individual di- and/or tri-substitutedadamantanes. EP 392 059, whose teaching is explicitly incorporatedherein by reference, is referred to for the production ofaminoadamantanes.

Aminoadamantane compounds have also already been used for pharmaceuticalpurposes. Thus, EP 392 059 discloses the use of 1-aminoadamantanecompounds for the treatment of Alzheimer's disease or brain cell damageas a result of cerebral ischemia. U.S. Pat. No. 3,450,761 describesaminoadamantanes with anti-viral activity.

Whether the activity of neutrophils is increased or partially orentirely inhibited depends on the concentration of the aminoadamantanecompound used. Thus, concentrations from 10⁻⁶ to 10⁻⁵ M have an activityincreasing effect. A concentration of about 5·10⁻⁶ M has provenespecially effective for increasing neutrophil activity—a concentrationwhich can be well achieved in vivo in blood plasma. In contrast, aninhibition of activity results with an increase of the aminoadamantaneconcentration. A concentration suitable for this purpose is 10⁻⁴ to 10⁻³M.

The invention comprises adamantane compounds substituted with one orwith more amino groups, wherein monoaminoadamantanes are preferred.Hence, suitable compounds of formula (I) are those wherein R₁ representsan amino group such as for example1-amino-3-ethyl-5,7-dimethyl-adamantane; as well as compounds wherein R₁represents an amino group and R₃ and R₄ represent a hydrogen atom, suchas for example 1-amino-3-cyclohexyl-adamantane or1-amino-3-ethyl-adamantane.

Preferred adamantane compounds are 1-amino-3,5-dimethyl-adamantane,1-amino-3,5-diethyl-adamantane and the N-substituted compounds1-N-methylamino-3,5-dimethyl-adamantane and the compound1-N-ethylamino-3,5-dimethyl-adamantane. Particularly preferred is1-amino-3,5-dimethyl-adamantane referred to as memantine (INN, AkatinolMemantine®) Görtelmeyer, R., et al, describe the treatment of Demenssyndrome with memantine in Spectrum of Neurorehabilitation, W.Zuckschwerdt Publishers, Munich, 1993, 50 ff.

As explained above, the aminoadamantane compounds according to theinvention only have a regulating effect on neutrophils which arepre-activated or have been stimulated by special activators.

Natural substances, such as Zymosan, N-formyl-Met-Leu-Phe (N-FLMP) or A23187 (a Ca antagonist) are known as activators. These substances can beused if one intends to specifically and more intensely activateneutrophils in vitro, but also in vivo. By this, it is possible todetect neutrophils in a sample, f or example in a sample of body fluid,especially a blood sample, even when only few neutrophils are present orwhen their activity is very weak. In this connection, the activator canbe present in combination with the aminoadamantane compound: The degreeof activation of the neutrophils stimulated by the activator isintensified by the simultaneous addition of aminoadamantane compound.

A further field of application is the improvement of immune defense inpersons with various forms of immune deficiencies, especially AIDSpatients. Here, a pharmaceutical composition can be administered whichcomprises a aminoadamantane compound according to the invention,optionally in combination with a neutrophil activator. Furthermore, theuse in CGD (agranulomatosis), Wegener's granulomatosis and/or glycogenstorage diseases is possible.

As a matter of course, the neutrophils can also be activated as aconsequence of a natural immune response of the organism as a reactionto diverse immunogens and trigger allergic reactions, inflammations andrheumatic symptoms due to overreaction. Here, the neutrophil activitycan be attenuated with suitably increased aminoadamantane concentrationsand the patient can be given relief. Internal and external inflammationconditions, for example in the knee, hip or jaw and also autoimmunediseases can be particularly combated effectively with aminoadamantanecontaining medicine. A further field of application is in the treatmentof parasitic diseases, such as Leishmannia.

In this connection, it is up to the discretion of the physician toassure the suitable concentration of the aminoadamantane compounds forthe regulation and modulation of neutrophil activity by selection of thesuitable dose and administration form. Depending on the field ofapplication, parenteral forms, for example intravenous or oraladministration forms are possible here; sustained action forms are alsosuitable. The invention also comprises combinations of theaminoadamantane compounds according to the invention as well aspharmaceutically acceptable salts, especially acid addition salts; forexample, hydrochlorides, hydrobromides, sulfates, acetates, succinates,tartrates or addition compounds with fumaric, maleic, citric orphosphoric acid are to be named here. The pharmaceutical compositionscan additionally contain a neutrophil activator depending on the rangeof indications. Customary pharmaceutically acceptable carriers andadjuvants are used for the formulation.

The following example illustrates the dependence of the activityincreasing or inhibiting effect of aminoadamantane derivatives by meansof the special compound, memantine.

EXAMPLE

Principle of Measurement

The determination of activity of stimulated neutrophils at variousmemantine concentrations was conducted by chemiluminescence measurement.Activated neutrophils form reactive oxygen species in the respiratoryburst which are detectable as very weak chemiluminescence—so-calledlow-level or ultra-weak chemiluminescence. To increase the photon yield,luminol is added as a sensitizer (indicator dependentchemiluminescence). Luminol is a cyclic hydrazide which can be oxidizedto diazoquinone through reactive oxygen species. By a nucleophil attackof a hydrogen peroxide anion, this diazoquinone is further converted toa-hydroxyhydroperoxide which disintegrates into aminophtalate underlight emission. Reaction batch:

Neutrophils: 10⁶ i.A. PBS-buffer pH (7.4): to 250 ml luminol: 80 mMmemantine: var. conc. stimulation agent: Zymosan 2.5 mg/ml or PMA 1 mMor A 23187 20 mM or N-FLMP 10 mM PBS = Phosphate Buffered Saline

Analogous reaction batches can be produced with further adamantanecompounds.

Measurement

Measurement was conducted with very sensitive, low-noisephotomultipliers which convert photons on a photocathode into primaryelectrons and then multiply amplify these to an electrical impulsesignal.

The reaction is started with a stimulation agent and measured withoutincubation time for 40 min (=80 cycles).

Results

With neutrophils which were activated with natural stimulation agents(Zymosan A; a23187; N-formyl-Met-Leu-Phe), the RLU-base rate (RLU=Relative Light Units) can be additionally increased with 10⁻⁶ Mmemantine (Zymosan A by about 90%; A 23187 by about 60%; N-FMLP by about78% of the base stimulation) [FIG. 2-4]. This suggests an increasedrespiratory burst and an increased phagocytosis activity. This reactiondoes not occur by stimulating with toxic Phorbol-Mystrate-Acetate (about−20% of the base stimulation) [FIG. 5]. In contrast, memantine almostcompletely inhibits the base stimulation of all stimulating agents at aconcentration of 10⁻³.

In a control experiment, it can be shown that memantine itself hasneither chemiluminescence nor quenches chemiluminescence (not shown).

Specificity of the Activation Regulation

The regulative effect of the aminoadamantane compounds according to theinvention is highly specific for neutrophils. Thus, the aminoadamantanecompounds do not show any action on the deregulation effect of activatedleukocytes. It emerges from FIG. 6 that ACC(1-aminocyclopropane-1-carboxylic acid) itself does not react withmemantine in relatively high concentrations. ACC is a specific indicatorfor myeloperoxidase which is released by degranulation of leukocytes andforms damaging hypochlorite in extracellular spaces. Myeloperoxidasereacts specifically with ACC under formation of ethylene which can bedetected gaschromatographically. Therewith, the formed amount ofethylene represents an indirect measuring quantity for leukocyteactivity.

In contrast, the indicator for the respiratory burst and the oxygenradicals arising therefrom (superoxide, OH-radicals andhydrogenperoxide) are activated by memantine. This is already shown at aconcentration of only 10⁻⁷ M which can easily be obtained in vivo. Theactivation effect can be made visible by addition of Fe³⁺ which shiftsthe equilibrium of the reaction through the Haber-Weiβ reaction fromsuperoxide and hydrogenperoxide to the direction of OH radicals. Asensitive indicator for oxygen species of this type is KMB(a-keto-g-methylthiobutyrate) which disintegrates into ethylene, amongothers, under oxidative conditions and which can be easily detected[FIG. 7].

As a control, FIG. 8 shows that the KMB-reaction, but not theACC-reaction, is stimulated first by the addition of Fe³⁺ at theconcentration of >5×10⁻⁴ M. Fe³⁺ alone is without influence.

What is claimed is:
 1. A process for increasing the activity of alreadyactivated neutrophils which comprises contacting said neutrophils withan aminoadamantane compound of formula (I)

wherein R₁, R₂, R₃ and R₄ are selected independently from each otherfrom —NR₅R₆, —NR₅R₆R₇ ⁺, hydrogen, aryl or heteroaryl with up to 7 ringmembers, C₁-C₂₀-alkyl, C₁-C₂₀-alkenyl and C₁-C₂₀-alkynyl, wherein thealkyl, alkenyl and alkynyl groups can be branched, unbranched orcyclized and optionally substituted with halogen, aryl or heteroarylwith up to 7 ring members with the proviso that at least one of thegroups R₁, R₂, R₃ and R₄ are represented by —NR₅R₆ or —NR₅R₆R₇ ⁺; andR₅, R₆ and R₇ are selected independently from each other from hydrogen,aryl or heteroaryl with up to 7 ring members, C₁-C₂₀-alkyl,C₁-C₂₀-alkenyl, C₁-C₂ -alkynyl, wherein the alkyl, alkenyl and alkynylgroups can be branched, unbranched or cyclized and optionallysubstituted with halogen, aryl or heteroaryl with up to 7 ring members,or R₅ and R₆, together with the nitrogen atom, form a heterocyclic groupwith up to 7 ring members in a concentration of said aminoadamantanecompound of from 10⁻⁶ to 10⁻⁵M.
 2. The process of claim 1, wherein theaminoadamantane compound of formula (I) is selected from the groupconsisting of 1-amino-3-ethyl-5,7-dimethyl-adamantane,1-amino-3-cyclohexyl-adamantane, 1-amino-3-ethyl-adamantane,1-amino-3,5-dimethyl-adamantane, 1-amino-3,5-diethyl-adamantane,1-N-methylamino-3,5-dimethyl-adamantane, and1-N-ethylamino-3,5-dimethyl-adamantane.
 3. The process of claim 1 or 2,wherein the activity of the neutrophils is increased in vitro.
 4. Theprocess of claim 1 or 2, for the treatment of CGD (agranulomatosis),Wegener's granulomatosis and/or immune deficiency diseases.
 5. Amedicament for increasing the activity of neutrophils comprising anaminoadamantane compound of formula (I);

wherein R₁, R₂, R₃ and R₄ are selected independently from each otherfrom —NR₅R₆, —NR₅R₆R₇ ⁺, hydrogen, aryl or heteroaryl with up to 7 ringmembers, C₁-C₂₀-alkyl, C₁-C₂₀-alkenyl and C₁-C₂₀-alkynyl, wherein thealkyl, alkenyl and alkynyl groups can be branched, unbranched orcyclized and optionally substituted with halogen, aryl or heteroarylwith up to 7 ring members with the proviso that at least one of thegroups R₁, R₂, R₃ and R₄ are represented by —NR₅R₆ or —NR₅R₆R₇ ⁺; andR₅, R₆ and R₇ are selected independently from each other from hydrogen,aryl or heteroaryl with up to 7 ring members, C₁-C₂₀-alkyl,C₁-C₂₀-alkenyl, C₁-C₂₀-alkynyl, wherein the alkyl, alkenyl and alkynylgroups can be branched, unbranched or cyclized and optionallysubstituted with halogen, aryl or heteroaryl with up to 7 ring members,or R₅ and R₆, together with the nitrogen atom, form a heterocyclic groupwith up to 7 ring members in a concentration of from 10⁻⁶ to 10⁻⁵M andan activator for neutrophils.
 6. A process for amplified, specificactivation of already activated neutrophils in vivo or in vitro whichcomprises contacting said neutrophils with a composition comprising anaminoadamantane compound formula (I)

wherein R₁, R₂, R₃ and R₄ are selected independently from each otherfrom —NR₅R₆, —NR₅R₆R₇ ⁺, hydrogen, aryl or heteroaryl with up to 7 ringmembers, C₁-C₂₀-alkyl, C₁-C₂₀-alkenyl and C₁-C₂₀-alkynyl, wherein thealkyl, alkenyl and alkynyl groups can be branched, unbranched orcyclized and optionally substituted with halogen, aryl or heteroryl withup to 7 ring members with the proviso that at least one of the groupsR₁, R₂, R₃ and R₄ are represented by —NR₅R₆ or —NR₅R₆R₇ ⁺; and R₅, R₆and R₇ are selected independently from each other from hydrogen, aryl orheteroaryl with up to 7 ring members, C₁-C₂₀-alkyl, C₁-C₂₀-alkenyl,C₁-C₂₀-alkynyl, wherein the alkyl, alkenyl and alkynyl groups can bebranched, unbranched or cyclized and optionally substituted withhalogen, aryl or heteroaryl with up to 7 ring members, or R₅ and R₆,together with the nitrogen atom, form a heterocyclic group with up to 7ring members in a concentration of from 10⁻⁶ to 10⁻⁵ M; and an activatorfor the neutrophils.
 7. The process of claim 6, wherein the neutrophilsare detected in a sample.
 8. The process of claim 6 or 7, wherein theactivator is selected from the groups consisting of Zymosan,N-formyl-Met-Leu-Phe and A
 23187. 9. The medicament of claim 5, whereinthe aminoadamantane compound of formula (I) is selected from the groupconsisting of 1-amino-3-ethyl-5,7-dimethyl-adamantane,1-amino-3-cyclohexyl-adamantane, 1-amino-3-ethyl-adamantane,1-amino-3,5-dimethyl-adamantane, 1-amino-3,5-diethyl-adamantane,1-N-methylamino-3,5-dimethyl-adamantane, and1-N-ethylamino-3,5-dimethyl-adamantane.
 10. The medicament of claim 5,wherein the activator is selected from the group consisting of Zymosan,N-formyl-Met-Leu-Phe and A 23187.